Investigation of Peptide Reactivity of Pro-hapten Skin Sensitizers Using a Peroxidase-Peroxide Oxidation System
Investigation of Peptide Reactivity of Pro-hapten Skin Sensitizers Using a Peroxidase-Peroxide Oxidation System
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DOI：
10.1093/toxsci/kfp192
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发表时间：
2009-11-01
影响因子：
3.8
通讯作者：
Lepoittevin, Jean-Pierre
中科院分区：
文献类型：
--
作者：
Gerberick, G. Frank;Troutman, John A.;Lepoittevin, Jean-Pierre
皮肤蛋白反应性是皮肤致敏发展中公认的关键步骤。了解化学物质与皮肤蛋白质反应或修饰皮肤蛋白质的能力与皮肤致敏性之间的关系，导致我们实验室开发了直接肽反应性测定（DPRA）。DPRA目前的局限性是它不能容易地测量前半抗原化学敏化剂的反应性。前半抗原是不直接反应的化学致敏剂，必须在体内生物活化以形成亲电子中间体。这项工作的结果表明，使用辣根过氧化物酶和过氧化氢（HRP/P）的评估前半抗原的皮肤致敏潜力的效用。与没有HRP/P的“直接”反应性评估相比，在与HRP/P共孵育后，观察到所检查的所有前半抗原的肽消耗的统计学显著增加。相反，在有和没有HRP/P的情况下，所有前半抗原的肽消耗百分比同样高（> 40%消耗），证明了自氧化途径。与此相反，肽耗尽所有nonsensitizing化学品检查是低的，没有HRP/P。最佳HRP/P浓度，孵育时间和最佳肽：化学品的比例进行了测定，使用一个敏感的和选择性的高效液相色谱串联质谱检测方法。掺入二硫苏糖醇以逆转含巯基的半胱氨酸肽亲核试剂的二聚化。这项初步工作显示了将酶介导的前半抗原活化步骤纳入化学皮肤致敏试验的潜力，该试验可检测所有类型的致敏剂。
Skin protein reactivity is a well established key step in the development of skin sensitization. Understanding the relationship between a chemical's ability to react with or modify skin protein and skin sensitization has led to the development of the Direct Peptide Reactivity Assay (DPRA) in our laboratory. A current limitation of the DPRA is that it cannot readily measure the reactivity of pro-hapten chemical sensitizers. Pro-haptens are chemical sensitizers that are not directly reactive and must be bioactivated in vivo to form an electrophilic intermediate(s). Results from this work demonstrate the utility of using horseradish peroxidase and hydrogen peroxide (HRP/P) for assessing the skin sensitization potential of pro-haptens. In comparison with "direct" reactivity assessments without HRP/P, statistically significant increases in peptide depletion for all pro-haptens examined were observed following coincubation with HRP/P. Conversely, the percent peptide depletion for all pre-haptens was equally high (> 40% depletion) with and without HRP/P demonstrating an auto-oxidation pathway. In contrast, peptide depletion for all nonsensitizing chemicals examined was low with and without HRP/P. The optimal HRP/P concentrations, incubation time and optimal peptide:chemical ratio were determined using a sensitive and selective high-performance liquid chromatography tandem mass spectrometry detection method. Dithiothreitol was incorporated to reverse the dimerization of the thiol-containing cysteine peptide nucleophile. This preliminary work shows the potential to incorporate an enzyme-mediated activation step for pro-haptens into an in chemico skin sensitization assay that results in the detection of all types of sensitizers.